Dysregulation of the MiR-30a-5p-B4GALT5 Axis Leads to Cell Proliferation and Is Associated with M1 Macrophage Polarization in PNH

Background：MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA's involvement in Paroxysmal nocturnal hemoglobinuria (PNH).

Methods: We constructed a PIGA knock-out K562 cell line and PIGA conditional knock-out mice model by CRISPR/cas9 technology. Furthermore, we used FACS and colony formation assays to clarify proliferative and apoptosis abilities of PNH cells. We also used FACS to clarify M1 Macrophage Polarization in mouse bone marrow-derived macrophages (BMDMs). RT-PCR, and western blot analyses were used to verify the relationship among miR-30a-5p, B4GALT5 and PI3K/AKT signaling.

Results: We observed that the expression of miR-30a-5p is significantly increased in Flaer negative cells compared with Flaer positive cells from 10 PNH patients (3.31±0.4 vs 1.48±0.23, p<0.05). The miRNA-mediated gene B4GALT5 were identified by comparing RNA-seq data with gene targets predicted using TargetScan. Importantly, B4GALT5 mRNA expression is significantly decreased in Flaer negative cells compared with Flaer positive cells from 15 PNH patients(1.68±0.31 vs 2.68±0.31, p<0.05), and this decreased B4GALT5 expression is also observed at the protein levels. In PIGA knock-out cells, the transfection of miR-30a-5p and B4GalT5 knockdown significantly promoted cell proliferation by activating PI3K/AKT signaling. What's more, the mean fluorescence intensity(MFI) of CD80 and CD86 which acted as the markers of M1 macrophages were higher in PNH patients compared with normal controls(CD80:1374±156 vs 614±162, p<0.05;CD86:738±131 vs 297±78,p<0.05). Meanwhile, downregulation of B4GalT5 increased the the markers of M1 macrophages in BMDMs of PIGA CKO mice.

Conclusion: Thus, miRNA-30a-5p/B4GALT5 axis plays a crucial role in modulating PNH clone proliferation and M1 macrophage Polarization. This is the first report to characterize the potential roles of miRNA-30a-5p in PNH.

Keywords: PNH, miRNA-30a-5p, B4GALT5, proliferation, Macrophage Polarization

No relevant conflicts of interest to declare.